This proposal is an extension of CA57629 that has been focused on understanding the metabolism, mechanism of action, and interaction of nucleoside analogs. With the success rate of analogs in leukemias, several laboratories including ours have investigated the mechanisms of cell death by these agents. The steps include formation of triphosphate of the analog, incorporation into replicating DNA, inhibition of ribonucleotide reductase (with newer analogs) and finally inhibition of DNA synthesis. Continued inhibition of DNA synthesis proceeds to cell death through apoptosis. When tested in cell lines, which are actively cycling and replicating DNA, such scenario seems to be in place. However, when one tries to validate this process during therapy, the outcome is conflicting and intriguing. The biology of leukemia cells in the body is very different from cell lines in culture. Leukemia cells in peripheral blood are generally non- or slow- cycling and with a very small percent of cells in S-phase (0-5%). Nonetheless after 5-days of effective nucleoside analog therapy, there is a massive cytoreduction (1 to 3-log decrease). Our hypothesis is built around these premises to suggest that in addition to conventional S-phase mediated pathway, there may he additional pathways that result in non-S-phase cell death during therapy. To test this hypothesis, we want to pursue three specific aims that are focused toward different mode of cell death by analogs. First, we plan to define the elements of cell death caused by conventional DNA synthesis inhibition pathway during therapy. Using nelarabine and clofarabine, two of the most successful new nucleoside analogs in the clinic, we plan to investigate the role of cellular pharmacokinetics and cellular pharmacodynamics in cell death. These parameters will be compared with clinical response to these therapies. Second, we plan to identify mitochondria induced cell death of leukemia cells during therapy. Nucleoside analogs may affect mitochondria directly and/or indirectly to induce cell death in circulating leukemia cells during therapy. Direct effect such as mitochondrial respiratory function involving ATP synthase, adenosine nucleotide translocator (ANT), and early decrease in mitochondnal membrane potential will be accessed to elucidate the role of mitochondria induced apoptosis. Indirect effect will include release of cytochrome c, and late effect on membrane potential. Finally, we will investigate the role of receptor-mediated cell death of leukemia cells during therapy. Following our lead in cell lines that analog incorporation results in induction of fas ligand followed by fas-mediated cell death of non-Sphase population, we plan to pursue the role of fas in cell death during therapy. We feel that knowledge gained through these aims will assist us in designing optimal therapy of leukemia with nucleoside analogs.